Metastasis remains responsible for over 90% of cancer-related deaths, and can be described as a series of physical events including tumor cell detachment from the primary site, invasion into the circulation, translocation through the blood to microvessels in distant organs, and transmigration through endothelium to form secondary tumors. One of the most common sites for metastasis is bone, which confers increased morbidity, a 5-year survival rate of ~25%, and median survival of ~40 months. Upon colonization in bone, tumor cells become highly resistant to chemotherapy, and effective treatments remain elusive. Bone marrow endothelial cells (BMECs) constitutively express the adhesion receptor E-selectin (ES) and the chemoattractant stromal cell-derived factor-1 (SDF-1), which bind to ES ligands (ESLs) and CXCR4 on tumor cells, respectively. Such interactions enable the adhesion and homing of tumor cells to BMECs necessary for tumor formation in bone. Down regulation or inhibition of ES and SDF-1 on BMECs provides a potential solution to disrupt bone metastasis formation. However, direct and specific inhibition of constitutive ES and SDF-1 on BMECs by small-molecules has been elusive, and blocking antibodies induce endothelial cell (EC) activation, which can promote further metastasis. Thus, ES and SDF-1 are promising candidates for combination RNA interference (RNAi) therapy, which inhibits traditionally undruggable targets by reducing mRNA expression. The challenge of utilizing siRNA is the need for safe, effective delivery methods, as unmodified siRNA is unstable in the bloodstream, immunogenic, and does not readily cross cell membranes. In the proposed research, we will develop and implement a bone marrow (BM) endothelium-targeted polymeric nanoparticle (NP) siRNA delivery platform to disrupt bone metastasis formation in vivo. Proven endothelium-specific siRNA delivery technology developed by our lab, termed 7C1, will now be functionalized with the targeting moiety Sialyl Lewis-X (SLeX) to enable targeted siRNA delivery and silencing of a target gene in BMECs in vivo, without reducing gene expression in other endothelium (Aim 1). We will conduct a screen to determine the most potent ES and SDF-1 siRNA candidates to encapsulate in targeted 7C1 (t-7C1), verify that t-7C1 can down regulate ES and SDF-1, and determine its efficacy in reducing tumor cell adhesion and transendothelial migration in vitro (Aim II). We will then determine the optimal siRNA dosage to down regulate ES and SDF-1 on BMECs in vivo, test the central hypothesis that down regulation of ES and SDF-1 will disrupt tumor cell adhesion and tumor formation in a mouse model of bone metastasis, and determine potential side effects of our approach on normal stem cell and leukocyte homing to BM (Aim III). The proposed project targets metastasis by disrupting the physical translocation of tumor cells to bone, and presents broadly enabling siRNA delivery technologies to silence multiple genes in BMECs that contribute to a range of diseases.